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液固界面上蛋白质迁移规律研究
【作者】 刘鹏；
【导师】 耿信笃；
【作者基本信息】 西北大学 ， 分析化学， 2009， 博士
【摘要】 蛋白质分离纯化是重组蛋白生产的瓶颈,高效液相色谱(high performance liquidchromatography,HPLC)是分离和纯化蛋白,特别是制备规模上的最有效的方法,也是制药工业中一种基本的分析和制备工具。蛋白质的性质与小分子不同,分离过程中,不仅会因分子大小的巨大差别,而且还会因分子构象发生的变化,使两者在色谱中保留行为存在很大差别。基于分离介质功能团和蛋白质的性质不同,优化分离操作参数是费时且耗财的过程。不同的样品中多肽,蛋白,小分子在色谱中的保留行为差别很大,所以,研究蛋白质在色谱中的保留行为规律对于色谱分离的条件优化和保留分子机理的研究有重要意义。本文研究内容分为5部分:文献综述:现有的描述蛋白色谱保留模型都是从小分子的色谱保留模型发展而来,在预测蛋白保留时间方面存在很大偏差。对现有的描述溶质色谱保留模型进行综述。共引用文献148篇,其中近三年的文献占到16%。以疏水色谱柱为例,研究蛋白质在疏水色谱(hydrophobic interactionchromatography,HIC)固定相界面上的迁移规律,对其保留时间和洗脱剂浓度、进样时间等之间关系进行研究。首次发现梯度洗脱中,蛋白质在分子构象基本不发生变化的条件下,其保留时间可分为不随进样时间变化的直线部分和随进样时间变化的开始迁移部分,并首次提出蛋白质在色谱中不连续迁移的概念。通过对该过程的自由能变化计算发现,对于同一根色谱柱,各种蛋白从开始迁移到完全洗脱的自由能变化为一定值,该自由能变化的大小与所使用的色谱柱有关。最后利用大分子在HIC中的保留行为规律,进行大小分子的分离和缓冲溶液交换理论研究。研究反相色谱(reversed phase liquid chromatography,RPLC)乙腈-水体系中,在蛋白质分子结构会发生变化,甚至变性的条件下,研究了流速对分离度和迁移的影响。结果发现:RPLC中小分子溶质的迁移速度随流速变化大,蛋白质类大分子溶质迁移随流速变化小。且溶质的相对保留值与流速之间存在双对数线性关系。研究了4根商品弱阴离子交换(weak-anion exchange chromatography,WAX)柱中存在的疏水相互作用机理。发现尽管存在的疏水作用强度不同,但是WAX中存在HIC机理是一个普遍现象。从分子机理上对该现象进行了解释,利用计量置换理论(stoichiometric displacement theory,SDT)对该现象进行了验证。利用WAX柱中存在HIC机理进行了在线单柱二维液相色谱分离纯化工业α-淀粉酶,并进行在线缓冲溶液交换。与单独的商品HIC柱进行比较发现,在线单柱二维分离纯化效果要远优于单独的HIC效果。
【Abstract】 The purity and separation of recombinant proteins are the technical bottleneck in down-stream technology of biotechnology. High performance liquid chromatography (HPLC) is the best way for purifying and separating the proteins, especially in preparative scale, it is also an important analytical tool in pharmaceutical industry. Due to the distinction of proteins and small molecular mass, the retention character of proteins during separation process by HPLC is quite different from that of small solutes. The changes in the molecular conformation of proteins during this process may change and bring in more difficulties. So it is important to optimize the separation parameter and to understand molecular mechanism of protein retention. Besides thermodynamic factors, the migration rule of solute involving in dynamic factors is the other important thing for this optimization. The thesis is to investigate the latter instance. The thesis contains five parts as follow:From the recent development of protein retention model was reviewed. When the retention mode of small solutes is employed for the prediction of protein retention, a great deviation occurs. This fact indicates that some problems scientists have net been found yet. 148 references were cited, the recent 3 years references account for 16%.Take hydrophobic interaction chromatography (HIC) column as an example, the migration rule of proteins on the interface between the stationary phase of HIC and mobile phase was investigated. It is firstly found that during gradients elution and the molecular conformation of proteins remains invariance, proteins retention is independent of the injection time until to a specific value for each protein. Thus, the retention of protein can be separated two parts: protein almost does not move as mobile phase strength is weaker than a certain value and protein starts to migrate until it is eluted out. In the latter instance, a quantitative relationship between the protein migration and the mobile phase composition was found. Based on this fact, a new concept of proteins discontinuous migration is firstly proposed.For reversed phase liquid chromatography (RPLC), using acetonitrile-water as mobile phase, when proteins were separated under the condition of molecular conformation of proteins changes, even denature, the foregoing discontinuous migration was also found. Small solutes were found to move faster than proteins when velocity of mobile phase increase, and also found a log-log linear relationship between relative retention time and flow-rate.Four commercial weak-anion exchange chromatography (WAX) columns were tested whether HIC retention exists, or not. The experimental result indicates that the existence of HIC mechanism in all of WAX column is a universal phenomenon, i.e., each of WAX column has a mixed-retention mode of WAX and HIC, although the extent is different. The phenomenon was explained by molecule mechanism and the stoichiometric displacement theory (SDT) was used to test this phenomenon.The HIC mechanism existed in WAX was used to on-line two-dimensional liquid chromatography using a single column (2DLC-1C) to separate industryα-amylase firstly, and buffer exchange was carried out also with on-line manner. Compared to commercial HIC, the purity efficiency of 2DLC-1C is superior to either a single HIC column, or a WAX IEC column.